Novel genetic products from ashbya gossypii, associated with the structure of the cell wall or the cytoskeleton

ABSTRACT

The invention relates to novel polynucleotides from  Ashbya gossypii;  to oligonucleotides hybridizing therewith; to expression cassettes and vectors which comprise these polynucleotides; to microorganisms transformed therewith; to polypeptides encoded by these polynucleotides; and to the use of the novel polypeptides and polynucleotides as targets for modulating the properties of the cell wall or of the cytoskeleton and, in particular, improving vitamin B2 production in microorganisms of the genus  Ashbya.

Novel gene products from Ashbya gossypii which are associated with theconstruction of the cell wall or of the cytoskeleton.

The present invention relates to novel polynucleotides from Ashbyagossypii; to oligonucleotides hybridizing therewith; to expressioncassettes and vectors which comprise these polynucleotides; tomicroorganisms transformed therewith; to polypeptides encoded by thesepolynucleotides; and to the use of the novel polypeptides andpolynucleotides as targets for modulating the properties of the cellwall or of the cytoskeleton and, in particular, improving vitamin B2production in microorganisms of the genus Ashbya.

Vitamin B2 (riboflavin, lactoflavin) is an alkali- and light-sensitivevitamin which shows a yellowish green fluorescence in solution. VitaminB2 deficiency may lead to ectodermal damage, in particular cataract,keratitis, corneal vascularization, or to autonomic and urogenitaldisorders. Vitamin B2 is a precursor for the molecules FAD and FMNwhich, besides NAD⁺ and NADP⁺, are important in biology for hydrogentransfer. They are formed from vitamin B2 by phosphorylation (FMN) andsubsequent adenylation (FAD).

Vitamin B2 is synthesized in plants, yeasts and many microorganisms fromGTP and ribulose 5-phosphate. The reaction pathway starts with openingof the imidazole ring of GTP and elimination of a phosphate residue.Deamination, reduction and elimination of the remaining phosphate resultin 5-amino-6-ribitylamino-2,4-pyrimidinone. Reaction of this compoundwith 3,4-dihydroxy-2-butanone 4-phosphate leads to the bicyclic molecule6,7-dimethyl-8-ribityllumazine. This compound is converted into thetricyclic compound riboflavin by dismutation, in which a 4-carbon unitis transferred.

Vitamin B2 occurs in many vegetables and in meat, and to a lesser extentin cereal products. The daily vitamin B2 requirement of an adult isabout 1.4 to 2 mg. The main breakdown product of the coenzymes FMN andFAD in humans is in turn riboflavin, which is excreted as such.

Vitamin B2 is thus an important dietary substance for humans andanimals. Efforts are therefore being made to make vitamin B2 availableon the industrial scale. It has therefore been proposed to synthesizevitamin B2 by a microbiological route. Microorganisms which can be usedfor this purpose are, for example, Bacillus subtilis, the ascomycetesEremothecium ashbyii, Ashbya gossypii, and the yeasts Candida flareriand Saccharomyces cerevisiae. The nutrient media used for this purposecomprise molasses or vegetable oils as carbon source, inorganic salts,amino acids, animal or vegetable peptones and proteins, and vitaminadditions. In sterile aerobic submerged processes, yields of more than10 g of vitamin B2 are obtained per liter of culture broth within a fewdays. The requirements are good aeration of the culture, carefulagitation and setting of temperatures below about 30° C. Removal of thebiomass, evaporation and drying of the concentrate result in a productenriched in vitamin B2.

Microbiological production of vitamin B2 is described, for example, inWO-A-92/01060, EP-A-0 405 370 and EP-A-0 531 708.

A survey of the importance, occurrence, production, biosynthesis and useof vitamin B2 is to be found, for example, in Ullmann's Encyclopaedia ofIndustrial Chemistry, volume A27, pages 521 et seq.

The cell wall and the cytoskeleton of a eukaryotic cell serve inparticular for maintaining the external and internal structure. Thefunctions of these components are comparable with those of a tent fabricand the relevant tent rods. Since, however, the cell framework of livingcells is not rigid but flexible and adaptable as required by growth andenvironmental conditions, the construction and the composition isinfluenced by external factors such as, for example, temperature and pH,but also by internal factors such as, for example, the ATP content orthe ion concentration of the cell.

The fungal cell wall plays a crucial part during the growth, developmentor interaction of the fungus with the environment and with other cells.Its primary function is protective, i.e. to protect the cell fromosmotic, chemical or biological damage. However, the cell wall is alsoinvolved in morphological responses, antigen expression, adhesion andcell-cell interaction. The fungal cell wall is composed of a mixture ofvarious polymers. Two categories are distinguished in this connection.Firstly, the so-called structural polymers which are responsible for therigidity of the structure and, secondly, the matrix polymers in whichthey are embedded, and which ensure a resistance to pressure. For mostfungi, the most important components of the cell wall are chitin,glucans and manno-proteins. Of these, chitin and glucans have structuralfunctions. Cell wall synthesis takes place by combining the individualcomponents in various stages. It is initially necessary for theindividual components to be synthesized inside the cell or at theplasmalemma/wall boundary layer. After all the polymers have beensecreted into the expanding wall, they initially form a loose assemblagevia molecular interactions before they are firmly linked together bycovalent bonds.

The cytoskeleton is by contrast a coordinated network of filamentouspolymers which are linked through various molecules to other cellularstructures. The organization and the properties of this network aresubject to a precise development-dependent and functional control. Themain structural components of the cytoskeleton are formed by the actinfilaments (F actin), microtubules and the intermediate filaments. Thecytosol may be compared more with a highly organized gel than with ahomogeneous solution, and the composition of the gel may show markeddifferences in different regions of the cell. The cytoskeletonundertakes important tasks in this structuring as well as in celldivision and organelle transport. In this connection it undertakes inthe metaphorical sense the function of railway tracks along which themost diverse cell components are moved by means of cell motors such asdynein or kinesin.

Construction of the cytoskeleton is, unlike the cell-wall structure, notcharacterized by the formation of covalent bonds. Since it must have aconsiderably greater flexibility, it is characterized as in the case ofthe microtubules by a “dynamic instability”. Tubulin subunits arepolymerized with the aid of GTP. Since, however, GTP has the property ofdecomposing to GDP+Pi under physiological conditions in the cell, thestructure of the microtubules is also weakened, so that they musttherefore be continuously synthesized in order to decompose againsubsequently. Microtubule-associated proteins (MAPs) make it possiblefor the cell to achieve greater or controllable stabilization of themicrotubules. MAPs have a high or low affinity, depending on the degreeof phosphorylation, and thus a controllable stabilizing effect onmicrotubules.

Polymerization of microfilaments from actin and regulation of thestability of these polymers in the cell takes place analogously to thatof tubulin. On the other hand, the process of polymerization is speededup by ATP. Actin-binding proteins influence the construction andbreakdown of the microfilaments and may, as in the case of profilin,even prevent actin polymerization.

During development-specific or environment-related change in themorphology of fungi, for example during budding or the development offruiting bodies or pseudohyphae there is extensive restructuring, whichis subject to extremely precise temporal and spatial regulation, bothduring cell wall synthesis and during cytoskeleton construction. Thebasic structural framework of the cell is essentially important for thestability of the cell and for vesicle transport and forms the basicrequirement for the production of biomass.

For a more detailed description of cell wall construction andcytoskeleton structuring, see Wessels, J. G. H. (1990), Role of cellwall architecture in fungal tip growth generation. In: Heath I. B. (ed)Tip growth in plant and fungal cells. Academic Press, San Diego, pp1-29; Heath I. B. and Heath M. C. (1978), Microtubules and organellemovement in the rust fungus Uromyces phaseoli var. Vignae. Cytobiologie16:393-411; McConnel S. J., Yaffe M. P. (1993), Intermediate filamentformation by a yeast protein essential for organelle inheritance.Science 260: 687-689; Esser K. und Lemke P. A. (ed) The Mycota—Acomprehensive Treatise on fungi as experimental systems for basic andapplied research. Springer-Verlag, Berlin; Voet D. und Voet J. G. (ed)Biochemie. VCH, Weinheim, and the references present in each of thesecitations.

The utilization of genes associated with the synthesis of the cell walland/or of the cytoskeleton for generating microorganisms, preferably ofthe genus Ashbya, in particular of Ashbya gossypii strains, withmodified cytoskeleton or modified cell wall and, for example, associatedtherewith a modified (higher) resistance to external effects has not yetbeen described.

It is an object of the present invention to provide novel targets forinfluencing the cell wall and cytoskeleton properties in microorganismsof the genus Ashbya, in particular in Ashbya gossypii. The object inparticular is to improve the stability of the cells in suchmicroorganisms. A further object is to improve the vitamin B2 productionby such microorganisms.

We have found that this object is achieved by providing encoding nucleicacid sequences which are upregulated in Ashbya gossypii during vitaminB2 production (based on results found with the aid of the MPSSanalytical method described in detail in the experimental part), and inparticular:

a) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a cell-wall precursor protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 8”.

In a further preferred embodiment of this aspect of the invention therehas been isolation according to the invention of a DNA clone which codesfor the full sequence of the nucleic acid of the invention and whichbears the internal name “Oligo 8v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 1. A furtheraspect of the invention relates-to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 4 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 8” and “Oligo 8v” have significant homologies withthe MIPS tag “Cwp1” from S. cerevisiae. The inserts have a nucleic acidsequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4. The amino acidsequence or amino acid part-sequence derived from the complementarystrand to SEQ ID NO: 1 or from the coding strand as shown in SEQ ID NO:4 has significant sequence homology with the cell-wall precursor proteinCwp1 from S. cerevisiae, described by Shimoni H., et al., in J. Biochem.118: 302-311 (1995).

b) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a serine-threonine kinase.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 25/39”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 25/39v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 8. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 10 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 25/39” and “Oligo 25/39v” have significanthomologies with the MIPS tag “ARK1” from S. cerevisiae. The inserts havea nucleic acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 10. Theamino acid sequence derived from the corresponding complementary strandto SEQ ID NO: 8 or from the coding strand as shown in SEQ ID NO: 10 hassignificant sequence homology with a serine-threonine protein kinasefrom S. cerevisiae.

c) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a GTPase-actiavting protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 46”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 46v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 12. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 15 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 46” and “Oligo 46v” have significant homologieswith the MIPS tag “BUD2/CLA2” from S. cerevisiae. The inserts have anucleic acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 15. Theamino acid sequence or amino acid part-sequence derived from thecorresponding complementary strand to SEQ ID NO: 12 or from the codingstrand as shown in SEQ ID NO: 15 has significant sequence homology witha GTPase-activating protein from S.cerevisiae, in particular homologywith the BUD2-encoded GTPase-activating protein for BUD2/Rsr1 describedby Park H.-O., et al., Nature 365: 269-274, (1993).

d) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of resistance to overexpression of actin.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 103”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 103v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 17. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 19 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 103” and “Oligo 103v” have significant homologieswith the MIPS tag “Aor1” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 19. The amino acidsequence or amino acid part-sequence derived from the correspondingcomplementary strand to SEQ ID NO: 17 or from the coding strand as shownin SEQ ID NO: 19 has significant sequence homology with a protein fromS. cerevisiae which has resistance to overexpression of actin orcontributes to this resistance.

e) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of an Nuf1p-like protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 128”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 128v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 21. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 23 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 128” and “Oligo 128v” have significant homologieswith the MIPS tag “Ykl179c” from S. cerevisiae. The inserts have anucleic acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23. Theamino acid sequence or amino acid part-sequence derived from the codingstrand has significant sequence homology with an Nuf1p-like protein fromS. cerevisiae. (cf. Wiemann S., et al., Yeast 9: 1343-1348 (1993)).

f) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of calponin or a calponin-homologousprotein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 150”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 150v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 26. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 28 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 150” and “Oligo 150v” have significant homologieswith the MIPS tag “Scp1” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 26 or SEQ ID NO: 28. The amino acidsequences derived in each case from the coding strand has significantsequence homology with a calponin or calponin-homologous protein from S.cerevisiae.

g) a, preferably upregulated, nucleic acid sequence which codes for aprotein which is essential for pseudohyphal development in Candidamaltosa.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 177”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 177v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 30. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 34. The polynucleotides can beisolated preferably from a microorganism of the genus Ashbya, inparticular A. gossypii. The invention additionally relates to thepolynucleotides complementary thereto; and to the sequences derived fromthese polynucleotides through the degeneracy of the genetic code.

The inserts of “Oligo 177” and “Oligo 177v” have significant homologieswith the MIPS tag “EPD1” from Candida maltosa. The inserts have anucleic acid sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 34. Aminoacid sequences which can be derived from the corresponding complementarystrand of SEQ ID NO: 30 or from the coding strand as shown in SEQ ID NO:34 have significant sequence homology with a protein from Candidamaltosa, in particular to a protein which is essential for pseudohyphaldevelopment in C. maltosa, (cf. Nakazawa T., et al., J. Bacteriol.,180(8), 2079-2086, (1998)). It was likewise possible to establishhomology to a corresponding protein from S. cerevisiae.

h) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of a protein which interacts with actin.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 145”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 145v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 36. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 38 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 145” and “Oligo 145v” have significant homologieswith the MIPS tag “Aip2” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 36 or SEQ ID NO: 38. The amino acidsequence or amino acid part-sequence derived from the coding strand hassignificant sequence homology with a protein from S. cerevisiae, whichinteracts with actin (cf. Chelstowska A., et al., Yeast 15 (13),1377-1391 (1999)).

A further aspect of the invention relates to oligonucleotides whichhybridize with one of the above polynucleotides, in particular understringent conditions.

The invention additionally relates to polynucleotides which hybridizewith one of the oligonucleotides of the invention and code for a geneproduct from microorganisms of the genus Ashbya or a functionalequivalent of this gene product.

The invention further relates to polypeptides or proteins which areencoded by the polynucleotides described above; and to peptide fragmentsthereof which have an amino acid sequence which comprises at least 10consecutive amino acid residues as shown in SEQ ID NO: 2, 3, 5, 6, 7, 9,11, 13, 14, 16, 18, 20, 22, 24, 25, 27, 29, 31, 32, 33, 35, 37 or SEQ IDNO: 39; and to functional equivalents of the polypeptides or proteins ofthe invention.

In this connection, functional equivalents differ from the productsspecifically disclosed in the invention by their amino acid sequencethrough addition, insertion, substitution, deletion or inversion at aminimum of one, such as, for example, 1 to 30 or 1 to 20 or 1 to 10,sequence positions without the originally observed protein function,which can be deduced by sequence comparison with other proteins, beinglost. It is thus possible for equivalents to have essentially identical,higher or lower activities compared with the native protein.

Further aspects of the invention relate to expression cassettes for therecombinant production of proteins of the invention, comprising one ofthe nucleic acid sequences defined above, operatively linked to at leastone regulatory nucleic acid sequence; and to recombinant vectorscomprising at least one such expression cassette of the invention.

Also provided according to the invention are prokaryotic or eukaryotichosts which are transformed with at least one vector of the above type.A preferred embodiment provides prokaryotic or eukaryotic hosts in whichthe functional expression of at least one gene which codes for apolypeptide of the invention as defined above is modulated (e.g.inhibited or overexpressed); or in which the biological activity of apolypeptide as defined above is reduced or increased. Preferred hostsare selected from ascomycetes, in particular those of the genus Ashbyaand preferably strains of A. gossypii.

Modulation of gene expression in the above sense includes bothinhibition thereof, for example through blockade of a stage inexpression (in particular transcription or translation) or a specificoverexpression of a gene (for example through modification of regulatorysequences or increasing the copy number of the coding sequence).

A further aspect of the invention relates to the use of an expressioncassette of the invention, of a vector of the invention or of a host ofthe invention for the microbiological production of vitamin B2 and/orprecursors and/or derivatives thereof.

A further aspect of the invention relates to the use of an expressioncassette of the invention, of a vector of the invention or of a host ofthe invention for the recombinant production of a polypeptide of theinvention as defined above.

Also provided according to the invention is a method for detecting orfor validating an effector target for modulating the microbiologicalproduction of vitamin B2 and/or precursors and/or derivatives thereof.This entails treating a microorganism capable of the microbiologicalproduction of vitamin B2 and/or precursors and/or derivatives thereofwith an effector which interacts with (such as, for example,non-covalently binds to) a target selected from a polypeptide of theinvention as defined above or a nucleic acid sequence coding therefor,validating the influence of the effector on the amount of themicrobiologically produced vitamin B2 and/or of the precursor and/or ofa derivative thereof; and isolating the target where appropriate. Thevalidation in this case takes place preferably by direct comparison withthe microbiological vitamin B2 production in the absence of the effectorunder otherwise identical conditions.

A further aspect of the invention relates to a method for modulating (inrelation to the amount and/or rate of) the microbiological production ofvitamin B2 and/or precursors and/or derivatives thereof, where amicroorganism capable of the microbiological production of vitamin B2and/or precursors and/or derivatives thereof is treated with an effectorwhich interacts with a target selected from a polypeptide of theinvention as defined above or a nucleic acid sequence coding therefor.

Preferred examples of the abovementioned effectors which should bementioned are:

-   -   a) antibodies or antigen-binding fragments thereof;    -   b) polypeptide ligands which are different from a) and which        interact with a polypeptide of the invention;    -   c) low molecular weight effectors which modulate the biological        activity of a polypeptide of the invention;    -   d) antisense nucleic acid sequences which interact with a        nucleic acid sequence of the invention.

The invention likewise relates to the abovementioned effectors havingspecificity for at least one of the targets, according to the invention,defined above.

A further aspect of the invention relates to a method for themicrobiological production of vitamin B2 and/or precursors and/orderivatives thereof, where a host as defined above is cultivated underconditions favoring the production of vitamin B2 and/or precursorsand/or derivatives thereof, and the desired product(s) is(are) isolatedfrom the culture mixture. It is preferred in this connection that thehost is treated with an effector as defined above before and/or duringthe cultivation. A preferred host is in this case selected frommicroorganisms of the genus Ashbya; in particular transformed asdescribed above.

A final aspect of the invention relates to the use of a polynucleotideor polypeptide of the invention as target for modulating the productionof vitamin B2 and/or precursors and/or derivatives thereof in amicroorganism of the genus Ashbya.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 1092 to595 in SEQ ID NO: 1) (upper sequence) and a part sequence of the MIPStag “Cwp1” from S. cerevisiae (lower sequence). Identical sequencepositions are indicated between the two sequences. Identical sequencepositions are indicated between the two sequences. Similar sequencepositions are labeled with “+”.

FIG. 2 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 1067 to84 in SEQ ID NO: 8) (upper sequence) and a part-sequence of the MIPS tagARK1 from S. cerevisiae (lower sequence). Identical sequence positionsare indicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 3A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 475 to353 in SEQ ID NO: 12) (upper sequence) and a part-sequence of the MIPStag BUD2/CLA2 from S. cerevisiae (lower sequence). FIG. 3B shows analignment between an amino acid part-sequence of the invention(corresponding to the complementary strand to position 351 to 1 in SEQID NO: 12) (upper sequence) and a part-sequence of the MIPS tagBUD2/CLA2 from S. cerevisiae (lower sequence). Identical sequencepositions are indicated between the two sequences. Similar sequencepositions are labeled with “+”.

FIG. 4 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 933 to157 in SEQ ID NO: 17) (upper sequence) and a part-sequence of the MIPStag Aor1 from S. cerevisiae (lower sequence). Identical sequencepositions are indicated between the two sequences. Similar sequencepositions are labeled with “+”.

FIG. 5 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand of position 117 to 794 in SEQ IDNO: 21) (upper sequence) and a part-sequence of the MIPS tag Ykl179cfrom S. cerevisiae (lower sequence). Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 6 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand to position 438 to 767 in SEQ IDNO: 26) (upper sequence) and a part-sequence of the MIPS tag Scp1 fromS. cerevisiae (lower sequence). Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 7A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 983 to651 in SEQ ID NO: 30) (upper sequence) and a part-sequence of the MIPStag EPD1 from C. maltosa (lower sequence). FIG. 7B shows an alignmentbetween an amino acid part-sequence of the invention (corresponding tothe complementary strand to position 661 to 596 in SEQ ID NO: 30) (uppersequence) and a part-sequence of the MIPS tag EPD1 from C. maltosa(lower sequence). FIG. 7C shows an alignment between an amino acidpart-sequence of the invention (corresponding to the complementarystrand to position 591 to 1 in SEQ ID NO: 30) (upper sequence) and apart-sequence of the MIPS tag EPD1 from C. maltosa (lower sequence).Identical sequence positions are indicated in each case between the twosequences. Similar sequence positions are labeled with “+”.

FIG. 8 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand in position 2 to 148 in SEQ IDNO: 36) (upper sequence) and a part-sequence of the MIPS tag Aip2 fromS. cerevisiae (lower sequence). Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid molecules of the invention encode polypeptides orproteins which are referred to here as proteins of the cell wall orcytoskeleton construction (for example with activity in relation to cellwall synthesis or cytoskeleton construction) or for short as “CCproteins”. These CC proteins have, for example, a function in thesynthesis or restructuring of cell wall or cytoskeleton for exampleassociated with development-specific or environment-related changes inthe morphology of the cell. Owing to the availability of cloning vectorswhich can be used in Ashbya gossypii, as disclosed, for example, inWright and Philipsen (1991) Gene, 109, 99-105, and of techniques forgenetic manipulation of A. gossypii and the related yeast species, thenucleic acid molecules of the invention can be used for geneticmanipulation of these organisms, in particular of A. gossypii, in orderto make them better and more efficient producers of vitamin B2 and/orprecursors and/or derivatives thereof. This improved production orefficiency may result from a direct effect of the manipulation of a geneof the invention or result from an indirect effect of such amanipulation.

The present invention is based on the provision of novel molecules whichare referred to here as CC nucleic acids and CC proteins and areinvolved in the construction of cell wall and cytoskeleton, inparticular in Ashbya gossypii (e.g. in the synthesis or restructuring ofcell wall and cytoskeleton). The activity of the CC molecules of theinvention in A. gossypii influences vitamin B2 production by thisorganism. The activity of the CC molecules of the invention ispreferably modulated so that the metabolic and/or energy pathways of A.gossypii in which the CC proteins of the invention are involved aremodulated in relation to the yield, production and/or efficiency ofvitamin B2 production, which modulates either directly or indirectly theyield, production and/or efficiency of vitamin B2 production in A.gossypii.

The nucleic acid sequences provided by the invention can be isolated,for example, from the genome of an Ashbya gossypii strain which isfreely available from the American Type Culture Collection under thenumber ATCC 10895.

Improvement in Vitamin B2 Production:

There is a number of possible mechanisms by which the yield, productionand/or efficiency of production of vitamin B2 by an A. gossypii straincan be influenced directly through changing the amount and/or activityof a CC protein of the invention.

Thus, a more efficient synthesis of cell wall and cytoskeleton may makethe cell more robust toward external influences so that the viabilityand thus the productivity in the fermenter is increased.

Mutagenesis of one or more CC proteins of the invention may also lead toCC proteins with altered (increased or reduced) activities whichinfluence indirectly the production of the required product from A.gossypii. It is possible, for example, with the aid of the CC proteinsto adapt the stability of the cells and vesicle transport in the cellsto the particular environmental or culturing conditions and thusmaintain the function of essential metabolic processes. These processesinclude besides the biosynthesis of the product also the construction ofthe cell walls, transcription, translation, biosynthesis of compoundswhich are necessary for the growth and division of cells (e.g.nucleotides, amino acids, vitamins, lipids etc.) (Lengeler et al.(1999)). By improving the growth and multiplication of these modifiedcells it is possible to increase the viability of the cells in cultureson the large scale and also to improve their rate of division so that acomparatively larger number of producing cells can survive in thefermenter culture. The yield, production or efficiency of production canbe increased at least because of the presence of a larger number ofviable cells each of which produces the required product.

Polypeptides

The invention relates to polypeptides which comprise the abovementionedamino acid sequences or characteristic part-sequences thereof and/or areencoded by the nucleic acid sequences described herein.

The invention likewise encompasses “functional equivalents” of thespecifically disclosed novel polypeptides.

“Functional equivalents” or analogs of the specifically disclosedpolypeptides are for the purposes of the present invention polypeptideswhich differ therefrom but which still have the desired biologicalactivity (such as, for example, substrate specificity).

“Functional equivalents” mean according to the invention in particularmutants which have in at least one of the abovementioned sequencepositions an amino acid which differs from that specifically mentionedbut nevertheless have one of the abovementioned biological activities.“Functional equivalents” thus comprise the mutants obtainable by one ormore amino acid additions, substitutions, deletions and/or inversions,it being possible for said modifications to occur in any sequenceposition as long as they lead to a mutant having the profile ofproperties of the invention. Functional equivalence exists in particularalso when there is qualitative agreement between mutant and unmodifiedpolypeptide in the reactivity pattern, i.e. there are differences in therate of conversion of identical substrates, for example.

“Functional equivalents” in the above sense are also precursors of thepolypeptides described, and functional derivatives and salts of thepolypeptides. The term “salts” means both salts of carboxyl groups andacid addition salts of amino groups in the protein molecules of theinvention. Salts of carboxyl groups can be prepared in a manner knownper se and comprise inorganic salts such as, for example, sodium,calcium, ammonium, iron and zinc salts, and salts with organic basessuch as, for example, amines such as triethanolamine, arginine, lysine,piperidine and the like. Acid addition salts such as, for example, saltswith mineral acids such as hydrochloric acid or sulfuric acid and saltswith organic acids such as acetic acid and oxalic acid are also anaspect of the invention.

“Functional derivatives” of polypeptides of the invention can also beprepared at functional amino acid side groups or at their N- orC-terminal end by known techniques. Such derivatives include for examplealiphatic esters of carboxyl groups, amides of carboxyl groupsobtainable by reaction with ammonia or with a primary or secondaryamine; N-acyl derivatives of free amino groups prepared by reaction withacyl groups; or O-acyl derivatives of free hydroxyl groups prepared byreaction with acyl groups.

“Functional equivalents” naturally also comprise polypeptides which areobtainable from other organisms, and naturally occurring variants. Forexample homologous sequence regions can be found by sequence comparison,and equivalent enzymes can be established on the basis of the specificrequirements of the invention.

“Functional equivalents” likewise comprise fragments, preferably singledomains or sequence motifs, of the polypeptides of the invention, whichhave, for example, the desired biological function.

“Functional equivalents” are additionally fusion proteins which have oneof the abovementioned polypeptide sequences or functional equivalentsderived therefrom and at least one other heterologous sequencefunctionally different therefrom in functional N- or C-terminal linkage(i.e. with negligible mutual impairment of the functions of the parts ofthe fusion proteins). Nonlimiting examples of such heterologoussequences are, for example, signal peptides, enzymes, immunoglobulins,surface antigens, receptors or receptor ligands.

“Functional equivalents” include according to the invention homologs ofthe specifically disclosed proteins. These have at least 60%, preferablyat least 75%, in particular at least 85%, such as, for example, 90%, 95%or 99%, homology to one of the specifically disclosed sequences,calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad,Sci. (USA) 85(8), 1988, 2444-2448.

In the case where protein glycosylation is possible, equivalents of theinvention include proteins of the type defined above in deglycosylatedor glycosylated form, and modified forms obtainable by altering theglycosylation pattern.

Homologs of the proteins or polypeptides of the invention can begenerated by mutagenesis, for example by point mutation or truncation ofthe protein. The term “homolog” as used here relates to a variant formof the protein which acts as agonist or antagonist of the proteinactivity.

Homologs of the proteins of the invention can be identified by screeningcombinatorial libraries of mutants such as, for example, truncationmutants. It is possible, for example, to generate a variegated libraryof protein variants by combinatorial mutagenesis at the nucleic acidlevel, such as, for example, by enzymatic ligation of a mixture ofsynthetic oligonucleotides. There is a large number of methods which canbe used to produce libraries of potential homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic gene can then be ligated into a suitable expression vector.The use of a degenerate set of genes makes it possible to provide allsequences which encode the desired set of potential protein sequences inone mixture. Methods for synthesizing degenerate oligonucleotides areknown to the skilled worker (for example Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidsRes. 11:477).

In addition, libraries of fragments of the protein codon can be used togenerate a variegated population of protein fragments for screening andfor subsequent selection of homologs of a protein of the invention. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double-stranded PCR fragment of a coding sequence with anuclease under conditions under which nicking takes place only aboutonce per molecule, denaturing the double-stranded DNA, renaturing theDNA to form double-stranded DNA, which may comprise sense/antisensepairs of different nicked products, removing single-stranded sectionsfrom newly formed duplices by treatment with S1 nuclease and ligatingthe resulting fragment library into an expression vector. It is possibleby this method to derive an expression library which encodes N-terminal,C-terminal and internal fragments having different sizes of the proteinof the invention.

Several techniques are known in the prior art for screening geneproducts from combinatorial libraries which have been produced by pointmutations or truncation and for screening cDNA libraries for geneproducts with a selected property. These techniques can be adapted torapid screening of gene libraries which have been generated bycombinatorial mutagenesis of homologs of the invention. The mostfrequently used techniques for screening large gene libraries undergoinghigh-throughput analysis comprise the cloning of the gene library intoreplicable expression vectors, transformation of suitable cells with theresulting vector library and expression of the combinatorial genes underconditions under which detection of the required activity facilitatesisolation of the vector which encodes the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening tests for identifying homologs(Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993)Protein Engineering 6(3):327-331).

Recombinant preparation of the polypeptides of the invention is possible(see following sections) or they can be isolated in the native form frommicroorganisms, especially those of the genus Ashbya, by use ofconventional biochemical techniques (see Cooper, T. G., BiochemischeArbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or inScopes, R., Protein Purification, Springer Verlag, New York, Heidelberg,Berlin).

Nucleic Acid Sequences:

The invention also relates to nucleic acid sequences (single- anddouble-stranded DNA and RNA sequences such as, for example, cDNA andmRNA), coding for one of the above polypeptides and their functionalequivalents which are obtainable, for example, by use of artificialnucleotide analogs.

The invention relates both to isolated nucleic acid molecules which codefor polypeptides or proteins of the invention or biologically activesections thereof, and to nucleic acid fragments which can be used, forexample, for use as hybridization probes or primers for identifying oramplifying coding nucleic acids of the invention.

The nucleic acid molecules of the invention may additionally compriseuntranslated sequences from the 3′ and/or 5′ end of the coding region ofthe gene.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acidand may moreover be essentially free of other cellular material orculture medium if it is produced by recombinant techniques, or free ofchemical precursors or other chemicals if it is chemically synthesized.

A nucleic acid molecule of the invention can be isolated by usingstandard techniques of molecular biology and the sequence informationprovided according to the invention. For example, cDNA can be isolatedfrom a suitable cDNA library by using one of the specifically disclosedcomplete sequences or a section thereof as hybridization probe andstandard hybridization techniques (as described, for example, inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). It ismoreover possible for a nucleic acid molecule comprising one of thedisclosed sequences or a section thereof to be isolated by polymerasechain reaction using the oligonucleotide primers constructed on thebasis of this sequence. The nucleic acid amplified in this way can becloned into a suitable vector and be characterized by DNA sequenceanalysis. The oligonucleotides of the invention which correspond to anSA nucleotide sequence can also be produced by standard syntheticmethods, for example using an automatic DNA synthesizer.

The invention additionally comprises the nucleic acid molecules whichare complementary to the specifically described nucleotide sequences, ora section thereof.

The nucleotide sequences of the invention make it possible to generateprobes and primers which can be used for identifying and/or cloninghomologous sequences in other cell types and organisms. Such probes andprimers usually comprise a nucleotide sequence region which hybridizesunder stringent conditions onto at least about 12, preferably at leastabout 25, such as, for example, 40, 50 or 75, consecutive nucleotides ofa sense strand of a nucleic acid sequence of the invention or acorresponding antisense strand.

Further nucleic acid sequences of the invention are derived from SEQ IDNO: 1, 4, 8, 10, 12, 15, 17, 19, 21, 23, 26, 28, 30, 34, 36 or SEQ IDNO: 38 and differ therefrom through addition, substitution, insertion ordeletion of one or more nucleotides, but still code for polypeptideshaving the desired profile of properties.

The invention also encompasses nucleic acid sequences which compriseso-called silent mutations or are modified, by comparison with aspecifically mentioned sequence, in accordance with the codon usage of aspecific source or host organism, as well as naturally occurringvariants, such as, for example, splice variants or allelic variants,thereof. It likewise relates to sequences which are obtainable byconservative nucleotide substitutions (i.e. the relevant amino acid isreplaced by an amino acid with the same charge, size, polarity and/orsolubility).

The invention also relates to molecules derived from the specificallydisclosed nucleic acids through sequence polymorphisms. These geneticpolymorphisms may exist because of the natural variation betweenindividuals within a population. These natural variations normallyresult in a variance of from 1 to 5% in the nucleotide sequence of agene.

The invention additionally encompasses nucleic acid sequences whichhybridize with or are complementary to the abovementioned codingsequences. These polynucleotides can be found on screening of genomic orcDNA libraries and, where appropriate, be amplified therefrom by meansof PCR using suitable primers, and then, for example, be isolated withsuitable probes. Another possibility is to transform suitablemicroorganisms with polynucleotides or vectors of the invention,multiply the microorganisms and thus the polynucleotides, and thenisolate them. An additional possibility is to synthesize polynucleotidesof the invention by chemical routes.

The property of being able to “hybridize” onto polynucleotides means theability of a polynucleotide or oligonucleotide to bind under stringentconditions to an almost complementary sequence, while there are nononspecific bindings between noncomplementary partners under theseconditions. For this purpose, the sequences should be 70-100%,preferably 90-100%, complementary. The property of complementarysequences being able to bind specifically to one another is made use of,for example, in the Northern or Southern blot technique or in PCR orRT-PCR in the case of primer binding. Oligonucleotides with a length of30 base pairs or more are normally employed for this purpose. Stringentconditions mean, for example, in the Northern blot technique the use ofa washing solution at 50-70° C., preferably 60-65° C., for example0.1×SSC buffer with 0.1% SDS (20×SSC: 3M NaCl, 0.3M Na citrate, pH 7.0)for eluting nonspecifically hybridized cDNA probes or oligonucleotides.In this case, as mentioned above, only nucleic acids with a high degreeof complementarity remain bound to one another. The setting up ofstringent conditions is known to the skilled worker and is described,for example, in Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

A further aspect of the invention relates to antisense nucleic acids.This comprises a nucleotide sequence which is complementary to a codingsense nucleic acid. The antisense nucleic acid may be complementary tothe entire coding strand or only to a section thereof. In a furtherembodiment, the antisense nucleic acid molecule is antisense to anoncoding region of the coding strand of a nucleotide sequence. The term“noncoding region” relates to the sequence sections which are referredto as 5′- and 3′-untranslated regions.

An antisense oligonucleotide may be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides long. An antisense nucleic acid ofthe invention can be constructed by chemical synthesis and enzymaticligation reactions using methods known in the art. An antisense nucleicacid can be synthesized chemically, using naturally occurringnucleotides or variously modified nucleotides which are configured sothat they increase the biological stability of the molecules or increasethe physical stability of the duplex formed between the antisense andsense nucleic acids. Examples which can be used are phosphorothioatederivatives and acridine-substituted nucleotides. Examples of modifiednucleosides which can be used for generating the antisense nucleic acidare, inter alia, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil,5-carboxy-methylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueuosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methyl-aminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queuosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,methyl uracil-5-oxyacetate, 3-(3-amino-3-carboxypropyl)uracil, (acp3)wand 2,6-diaminopurine. The antisense nucleic acid may also be producedbiologically by using an expression vector into which a nucleic acid hasbeen subcloned in the antisense direction.

The antisense nucleic acid molecules of the invention are normallyadministered to a cell or generated in situ so that they hybridize withthe cellular mRNA and/or a coding DNA or bind thereto, so thatexpression of the protein is inhibited for example by inhibition oftranscription and/or translation.

The antisense molecule can be modified so that it binds specifically toa receptor or to an antigen which is expressed on a selected cellsurface, for example through linkage of the antisense nucleic acidmolecule to a peptide or an antibody which binds to a cell surfacereceptor or antigen. The antisense nucleic acid molecule can also beadministered to cells by using the vectors described herein. The vectorconstructs preferred for achieving adequate intracellular concentrationsof the antisense molecules are those in which the antisense nucleic acidmolecule is under the control of a strong bacterial, viral or eukaryoticpromoter.

In a further embodiment, the antisense nucleic acid molecule of theinvention is an alpha-anomeric nucleic acid molecule. An alpha-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA, with the strands running parallel to one another, incontrast to normal alpha units (Gaultier et al., (1987) Nucleic AcidsRes. 15:6625-6641). The antisense nucleic acid molecule may additionallycomprise a 2′-O-methylribonucleotide (Inoue et al., (1987) Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987)FEBS Lett. 215:327-330).

The invention also relates to ribozymes. These are catalytic RNAmolecules with ribonuclease activity which are able to cleave asingle-stranded nucleic acid such as an mRNA to which they have acomplementary region. It is thus possible to use ribozymes (for examplehammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) for the catalytic cleavage of transcripts of the inventionin order thereby to inhibit the translation of the corresponding nucleicacid. A ribozyme with specificity for a coding nucleic acid of theinvention can be formed, for example, on the basis of a cDNAspecifically disclosed herein. For example a derivative of atetrahymena-L-19 IVS RNA can be constructed, with the nucleotidesequence of the active site being complementary to the nucleotidesequence to be cleaved in a coding mRNA of the invention. (Compare, forexample, U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742).Alternatively, mRNA can be used for selecting a catalytic RNA withspecific ribonuclease activity from a pool of RNA molecules (see, forexample, Bartel, D., and Szostak, J. W. (1993) Science 261:1411-1418).

Gene expression of sequences of the invention can alternatively beinhibited by targeting nucleotide sequences which are complementary tothe regulatory region of a nucleotide sequence of the invention (forexample to a promoter and/or enhancer of a coding sequence) so thatthere is formation of triple helix structures which preventtranscription of the corresponding gene in target cells (Helene, C.(1991) Anticancer Drug Res. 6(6) 569-584; Helene, C. et al., (1992) Ann.N. Y. Acad. Sci. 660:27-36; and Maher., L. J. (1992) Bioassays14(12):807-815).

Expression Constructs and Vectors:

The invention additionally relates to expression constructs comprising,under the genetic control of regulatory nucleic acid sequences, anucleic acid sequence coding for a polypeptide of the invention; and tovectors comprising at least one of these expression constructs. Suchconstructs of the invention preferably comprise a promoter 5′-upstreamfrom the particular coding sequence, and a terminator sequence3′-downstream, and, where appropriate, other usual regulatory elements,in particular each operatively linked to the coding sequence. “Operativelinkage” means the sequential arrangement of promoter, coding sequence,terminator and, where appropriate, other regulatory elements in such away that each of the regulatory elements is able to comply with itsfunction as intended for expression of the coding sequence. Examples ofsequences which can be operatively linked are targeting sequences andenhancers, polyadenylation signals and the like. Other regulatoryelements comprise selectable markers, amplification signals, origins ofreplication and the like. Suitable regulatory sequences are described,for example, in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).

In addition to the artificial regulatory sequences it is possible forthe natural regulatory sequence still to be present in front of theactual structural gene. This natural regulation can, where appropriate,be switched off by genetic modification, and expression of the genes canbe increased or decreased. The gene construct can, however, also have asimpler structure, that is to say no additional regulatory signals areinserted in front of the structural gene, and the natural promoter withits regulation is not deleted. Instead, the natural regulatory sequenceis mutated so that regulation no longer takes place, and gene expressionis enhanced or diminished. The nucleic acid sequences may be present inone or more copies in the gene construct.

Examples of promoters which can be used are: cos, tac, trp, tet,trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PRor λ-PL promoter, which are advantageously used in Gram-negativebacteria; and the Gram-positive promoters amy and SPO2, the yeastpromoters ADC1, MF□, AC, P-60, CYC1, GAPDH or the plant promotersCaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, not or the ubiquitin orphaseolin promoter. The use of inducible promoters is particularlypreferred, such as, for example, light- and, in particular,temperature-inducible promoters such as the P_(r)P_(l) promoter. It ispossible in principle for all natural promoters with their regulatorysequences to be used. In addition, it is also possible advantageously touse synthetic promoters.

Said regulatory sequences are intended to make specific expression ofthe nucleic acid sequences possible. This may mean, for example,depending on the host organism, that the gene is expressed oroverexpressed only after induction or that it is immediately expressedand/or overexpressed.

The regulatory sequences or factors may moreover preferably influencepositively, and thus increase or reduce, expression. Thus, enhancementof the regulatory elements can take place advantageously at the level oftranscription by using strong transcription signals such as promotersand/or enhancers. However, it is also possible to enhance translationby, for example, improving the stability of the mRNA.

An expression cassette is produced by fusing a suitable promoter to asuitable nucleotide sequence of the invention and to a terminator signalor polyadenylation signal. Conventional techniques of recombination andcloning are used for this purpose, as described, for example, in T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1984) and in Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley lnterscience (1987).

For expression in a suitable host organism, the recombinant nucleic acidconstruct or gene construct is advantageously inserted into ahost-specific vector, which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and can befound, for example, in “Cloning Vectors” (Pouwels P. H. et al., eds,Elsevier, Amsterdam-New York-Oxford, 1985). Vectors also mean not onlyplasmids but also all other vectors known to the skilled worker, suchas, for example, phages, viruses, such as SV40, CMV, baculovirus andadenovirus, transposons, IS elements, phasmids, cosmids, and linear orcircular DNA. These vectors may undergo autonomous replication in thehost organism or chromosomal replication.

Examples of suitable expression vectors which may be mentioned are:

Conventional fusion expression vectors such as pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), with which respectively glutathione S-transferase (GST), maltoseE-binding protein and protein A are fused to the recombinant targetprotein.

Non-fusion protein expression vectors such as pTrc (Amann et al., (1988)Gene 69:301-315) and pET 11d (Studier et al. Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)60-89).

Yeast expression vector for expression in the yeast S. cerevisiae, suchas pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFα (Kurjan andHerskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for constructing vectors suitable for the use inother fungi such as filamentous fungi comprise those which are describedin detail in: van den Hondel, C.A.M.J.J. & Punt, P. J. (1991) “Genetransfer systems and vector development for filamentous fungi, in:Applied Molecular Genetics of Fungi, J. F. Peberdy et al., eds, pp.1-28,Cambridge University Press: Cambridge.

Baculovirus vectors which are available for expression of proteins incultured insect cells (for example Sf9 cells) comprise the pAc series(Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and pVL series(Lucklow and Summers (1989) Virology 170:31-39).

Plant expression vectors such as those described in detail in: Becker,D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformations”, Nucl. Acids Res.12:8711-8721.

Mammalian expression vectors such as pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).

Further suitable expression systems for prokaryotic and eukaryotic cellsare described in chapters 16 and 17 of Sambrook, J., Fritsch, E. F. andManiatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Recombinant Microorganisms:

The vectors of the invention can be used to produce recombinantmicroorganisms which are transformed, for example, with at least onevector of the invention and can be employed for producing thepolypeptides of the invention. The recombinant constructs of theinvention described above are advantageously introduced and expressed ina suitable host system. Cloning and transfection methods familiar to theskilled worker, such as, for example, coprecipitation, protoplastfusion, electroporation, retroviral transfection and the like, arepreferably used to bring about expression of said nucleic acids in theparticular expression system. Suitable systems are described, forexample, in Current Protocols in Molecular Biology, F. Ausubel et al.,eds, Wiley Interscience, New York 1997, or Sambrook et al. MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

It is also possible according to the invention to produce homologouslyrecombined microorganisms. This entails production of a vector whichcontains at least one section of a gene of the invention or a codingsequence, in which, where appropriate, at least one amino acid deletion,addition or substitution has been introduced in order to modify, forexample functionally disrupt, the sequence of the invention (knockoutvector). The introduced sequence may, for example, also be a homologfrom a related microorganism or be derived from a mammalian, yeast orinsect source. The vector used for homologous recombination mayalternatively be designed so that the endogenous gene is mutated orotherwise modified during the homologous recombination but still encodesthe functional protein (for example the regulatory region locatedupstream may be modified in such a way that this modifies expression ofthe endogenous protein). The modified section of the CC gene is in thehomologous recombination vector. The construction of suitable vectorsfor homologous recombination is, for example, described in Thomas, K. R.and Capecchi, M. R. (1987) Cell 51:503.

Suitable host organisms are in principle all organisms which enableexpression of the nucleic acids of the invention, their allelicvariants, their functional equivalents or derivatives. Host organismsmean, for example, bacteria, fungi, yeasts, plant or animal cells.Preferred organisms are bacteria, such as those of the generaEscherichia, such as, for example, Escherichia coli, Streptomyces,Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomycescerevisiae, Aspergillus, higher eukaryotic cells from animals or plants,for example Sf9 or CHO cells. Preferred organisms are selected from thegenus Ashbya, in particular from A. gossypii strains.

Successfully transformed organisms can be selected through marker geneswhich are likewise present in the vector or in the expression cassette.Examples of such marker genes are genes for antibiotic resistance andfor enzymes which catalyze a color-forming reaction which causesstaining of the transformed cell. These can then be selected byautomatic cell sorting. Microorganisms which have been successfullytransformed with a vector and harbor an appropriate antibioticresistance gene (for example G418 or hygromycin) can be selected byappropriate antibiotic-containing media or nutrient media. Markerproteins present on the surface of the cell can be used for selection bymeans of affinity chromatography.

The combination of the host organisms and the vectors appropriate forthe organisms, such as plasmids, viruses or phages, such as, forexample, plasmids with the RNA polymerase/promoter system, phages λ or μor other temperate phages or transposons and/or other advantageousregulatory sequences forms an expression system. The term “expressionsystem” means, for example, the combination of mammalian cells, such asCHO cells, and vectors, such as pcDNA3neo vector, which are suitable formammalian cells.

If desired, the gene product can also be expressed in transgenicorganisms such as transgenic animals such as, in particular, mice, sheepor transgenic plants.

Recombinant Production of the Polypeptides:

The invention further relates to methods for the recombinant productionof a polypeptide of the invention or functional, biologically activefragments thereof, wherein a polypeptide-producing microorganism iscultured, expression of the polypeptides is induced where appropriate,and they are isolated from the culture. The polypeptides can also beproduced on the industrial scale in this way if desired.

The recombinant microorganism can be cultured and fermented by knownmethods. Bacteria can be grown, for example, in TB or LB medium and at atemperature of 20 to 40° C. and a pH of from 6 to 9. Details of suitableculturing conditions are described, for example, in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).

If the polypeptides are not secreted into the culture medium, the cellsare then disrupted and the product is obtained from the lysate by knownprotein isolation methods. The cells may alternatively be disrupted byhigh-frequency ultrasound, by high pressure, such as, for example, in aFrench pressure cell, by osmolysis, by the action of detergents, lyticenzymes or organic solvents, by homogenizers or by a combination of aplurality of the methods mentioned.

The polypeptides can be purified by known chromatographic methods suchas molecular sieve chromatography (gel filtration), such as Q-Sepharosechromatography, ion exchange chromatography and hydrophobicchromatography, and by other usual methods such as ultrafiltration,crystallization, salting out, dialysis and native gel electrophoresis.Suitable methods are described, for example, in Cooper, T. G.,Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New Yorkor in Scopes, R., Protein Purification, Springer Verlag, New York,Heidelberg, Berlin.

It is particularly advantageous for isolation of the recombinant proteinto use vector systems or oligonucleotides which extend the cDNA byparticular nucleotide sequences and thus code for modified polypeptidesor fusion proteins which serve, for example, for simpler purification.Suitable modifications of this type are, for example, so-called tagswhich act as anchors, such as, for example, the modification known ashexa-histidine anchor, or epitopes which can be recognized as antigensby antibodies (described, for example, in Harlow, E. and Lane, D., 1988,Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Theseanchors can be used to attach the proteins to a solid support, such as,for example, a polymer matrix, which can, for example, be packed into achromatography column, or can be used on a microtiter plate or anothersupport.

These anchors can at the same time also be used for recognition of theproteins. It is also possible to use for recognition of the proteinsconventional markers such as fluorescent dyes, enzyme markers which forma detectable reaction product after reaction with a substrate, orradioactive labels, alone or in combination with the anchors forderivatizing the proteins.

The invention additionally relates to a method for the microbiologicalproduction of vitamin B2 and/or precursors and/or derivatives thereof.

If the conversion is carried out with a recombinant microorganism, themicroorganisms are preferably initially cultured in the presence ofoxygen and in a complex medium, such as, for example, at a culturingtemperature of about 20° C. or more, and at a pH of about 6 to 9 untilan adequate cell density is reached. In order to be able to control thereaction better, it is preferred to use an inducible promoter. Theculturing is continued in the presence of oxygen for 12 hours to 3 daysafter induction of vitamin B2 production.

The following nonlimiting examples describe specific embodiments of theinvention.

General Experimental Details

a) General Cloning Methods

The cloning steps carried out for the purpose of the present invention,such as, for example, restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of E. coli cells, culturing of bacteria, replication ofphages and sequence analysis of recombinant DNA, were carried out asdescribed by Sambrook et al. (1989) loc. cit.

b) Polymerase Chain Reaction (PCR)

PCR was carried out in accordance with a standard protocol with thefollowing standard mixture:

8 μl of dNTP mix (200 μM), 10 μl of Taq polymerase buffer (10×) withoutMgCl₂, 8 μl of MgCl₂ (25 mM), 1 μl of each primer (0.1 μM), 1 μl of DNAto be amplified, 2.5 U of Taq polymerase (MBI Fermentas, Vilnius,Lithuania), demineralized water ad 100 μl.

c) Culturing of E. coli

The recombinant E. coli DH5α strain was cultured in LB-amp medium(tryptone 10.0 g, NaCl 5.0 g, yeast extract 5.0 g, ampicillin 100 g/ml,H₂O ad 1000 ml) at 37° C. For this purpose, in each case one colony wastransferred, using an inoculating loop, from an agar plate into 5 ml ofLB-amp. After culturing for about 18 hours shaking at a frequency of 220rpm, 400 ml of medium in a 2 l flask were inoculated with 4 ml ofculture.

Induction of P450 expression in E. coli took place after the OD578reached a value between 0.8 and 1.0 by heat-shock induction at 42° C.for three to four hours.

d) Purification of the Required Product from the Culture

The required product can be isolated from the microorganism or from theculture supernatant by various methods known in the art. If the requiredproduct is not secreted by the cells, the cells can be harvested fromthe culture by slow centrifugation, and the cells can be lysed bystandard techniques such as mechanical force or ultrasound treatment.

The cell detritus is removed by centrifugation, and the supernatantfraction which contains the soluble proteins is obtained for furtherpurification of the required compound. If the product is secreted by thecells, the cells are removed from the culture by slow centrifugation,and the supernatant fraction is retained for further purification.

The supernatant fraction from the two purification methods is subjectedto a chromatography with a suitable resin, with the required moleculeeither being retained on the chromatography resin, or passing throughthe latter, with greater selectivity than the impurities. Thesechromatography steps can be repeated if necessary, using the same ordifferent chromatography resins. The skilled worker is proficient in theselection of suitable chromatography resins and their most effective usefor a particular molecule to be purified. The purified product can beconcentrated by filtration or ultrafiltration and be stored at atemperature at which the stability of the product is maximal.

Many purification methods are known in the art. These purificationtechniques are described, for example, in Bailey, J. E. & Ollis, D. F.Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined byprior art techniques. These comprise high performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzyme assay or microbiological assays.These analytical methods are summarized in: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH:Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, pp. 575-581and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 17.

e) General Description of the MPSS Method, Clone Identification andHomology Search

The MPSS technology (Massive Parallel Signature Sequencing as describedby Brenner et al, Nat. Biotechnol. (2000) 18, 630-634; to which expressreference is hereby made) was applied to the filamentous, vitaminB2-producing fungus Ashbya gossypii. It is possible with the aid of thistechnology to obtain with high accuracy quantitative information aboutthe level of expression of a large number of genes in a eukaryoticorganism. This entails the mRNA of the organism being isolated at aparticular time X, being transcribed with the aid of the enzyme reversetranscriptase into cDNA and then being cloned into special vectors whichhave a specific tag sequence. The number of vectors with a different tagsequence is chosen to be high enough (about 1000 times higher) forstatistically each DNA molecule to be cloned into a vector which isunique through its tag sequence.

The vector inserts are then cut out together with the tag. The DNAmolecules obtained in this way are then incubated with microbeads whichpossess the molecular counterparts of the tags mentioned. Afterincubation it can be assumed that each microbead is loaded via thespecific tags or counterparts with only one type of DNA molecules. Thebeads are transferred into a special flow cell and fixed there so thatit is possible to carry out a mass sequencing of all the beads with theaid of an adapted sequencing method based on fluorescent dyes and withthe aid of a digital color camera. Although numerically high analysis ispossible with this method, it is limited by a reading width of about 16to 20 base pairs. The sequence length is, however, sufficient to make anunambiguous correlation between sequence and gene possible for mostorganisms (20 bp have a sequence frequency of ˜1×10¹²; compared withthis, the human genome has a size of “only” ˜3×10⁹ bp).

The data obtained in this way are analyzed by counting the number ofidentical sequences and comparing their frequencies with one another.Frequently occurring sequences reflect a high level of expression, andsequences which occur singly a low level of expression. If the mRNA wasisolated at two different time points (X and Y), it is possible toconstruct a chronological expression pattern of individual genes.

EXAMPLE 1

Isolation of mRNA from Ashbya gossypii

Ashbya gossypii was cultured in a manner known per se (nutrient medium:27.5 g/l yeast extract; 0.5 g/l magnesium sulfate; 50 ml/I soybean oil;pH 7). Ashbya gossypii mycelium samples are taken at various timesduring the fermentation (24 h, 48 h and 72 h), and the corresponding RNAor mRNA is isolated therefrom according to the protocol of Sambrook etal. (1989).

EXAMPLE 2

Application of the MPSS

Isolated mRNA from A. gossypii is then subjected to an MPSS analysis asexplained above.

The sets of data found are subjected to a statistical analysis andcategorized according to the significance of the differences inexpression. This entailed examination both in relation to an increaseand a reduction in the level of expression. A division is made byclassifying the change in expression into a) monotonic change, b) changeafter 24 h, and c) change after 48 h.

The 20 bp sequences representing a change in expression and found byMPSS analysis are then used as probes and hybridized with a gene libraryfrom Ashbya gossypii, with an average insert size of about 1 kb. Thehybridization temperature in this case was in the range from about 30 to57° C.

EXAMPLE 3

Construction of a Genomic Gene Library from Ashbya gossypii

To construct a genomic DNA library, initially chromosomal DNA isisolated by the method of Wright and Philippsen (Gene (1991) 109:99-105) and Mohr (1995, PhD Thesis, Biozentrum Universität Basel,Switzerland).

The DNA is partially digested with Sau3A. For this purpose, 6 μg ofgenomic DNA are subjected to a Sau3A digestion with various amounts ofenzyme (0.1 to 1 U). The fragments are fractionated in a sucrose densitygradient. The 1 kb region is isolated and subjected to a QiaExextraction. The largest fragments are ligated to the BamHl-cut vectorpRS416 (Sikorski and Hieter, Genetics (1988) 122; 19-27) (90 ng ofBamHl-cut, dephosphorylated vector; 198 ng of insert DNA; 5 ml of water;2 μl of 10× ligation buffer; 1 U ligase). This ligation mixture is usedto transform the E. coli laboratory strain XL-1 blue, and the resultingclones are employed for identifying the insert.

EXAMPLE 4

Preparation of an Ordered Gene Library (CHIP Technology)

About 25,000 colonies of the Ashbya gossypii gene library (thiscorresponds to approximately a 3-fold coverage of the genome) weretransferred in an ordered manner to a nylon membrane and then treated bythe method of colony hybridization as described in Sambrook et al.(1989). Oligonucleotides were synthesized from the 20 bp sequences foundby MPSS analysis and were radiolabeled with ³²P. In each case 10 labeledoligonucleotides with a similar melting point are combined andhybridized together with the nylon membranes. After hybridization andwashing steps, positive clones are identified by autoradiography andanalyzed directly by PCR sequencing.

In this way, a clone which harbors an insert with the internal name“Oligo 8” and has significant homologies with the MIPS tag “Cwp1” fromS. cerevisiae was identified. The insert has a nucleic acid sequence asshown in SEQ ID NO: 1.

In this way, a further clone which harbors an insert with the internalname “Oligo 25/39” and has significant homologies with the MIPS tag“ARK1” from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 8.

In this way, a further clone which harbors an insert with the internalname “Oligo 46” and has significant homologies with the MIPS tag“BUD2/CLA2” from S. cerevisiae was identified. The insert has a nucleicacid sequence as shown in SEQ ID NO: 12.

In this way, a further clone which harbors an insert with the internalname “Oligo 103” and has significant homologies with the MIPS tag “Aor1”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 17.

In this way, a further clone which harbors an insert with the internalname “Oligo 128” and has significant homologies with the MIPS tag“Ykl179c” from S. cerevisiae was identified. The insert has a nucleicacid sequence as shown in SEQ ID NO: 21.

In this way, a further clone which harbors an insert with the internalname “Oligo 150” and has significant homologies with the MIPS tag “Scp1”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 26.

In this way, a clone which harbors an insert with the internal name“Oligo 177” and has significant homologies with the MIPS tag “EPD1” fromC. maltosa was identified. The insert has a nucleic acid sequence asshown in SEQ ID NO: 30.

In this way, a clone which harbors an insert with the internal name“Oligo 145” and has significant homologies with the MIPS tag “Aip 2”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 36.

EXAMPLE 5

Analysis of the Sequence Data by Means of a BLASTX Search

An analysis of the resulting nucleic acid sequences, i.e. theirfunctional assignment to a functional amino acid sequence took place bymeans of a BLASTX search in sequence databases. Almost all of the aminoacid sequence homologies found related to Saccharomyces cerevisiae(baker's yeast). Since this organism had already been completelysequenced, more detailed information about these genes could be referredto under:

-   -   http://www.mips.gsf.de/proj/yeast/search/code_search.htm.

Thus, the following homologies with an amino acid fragment from S.cerevisiae were found. The corresponding alignments are shown in FIGS. 1to 8 which are appended.

a) The amino acid sequence derived from the coding strand in SEQ ID NO:1has significant sequence homology with a cell-wall precursor proteinfrom S. cerevisiae. An amino acid part-sequence derived therefrom(corresponding to nucleotides 1092 to 595 from SEQ ID NO:1) with apart-sequence of the S. cerevisiae protein is depicted in FIG. 1. SEQ IDNO: 2 and SEQ ID NO: 3 in each case show an N-terminally extended aminoacid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a cell-wall precursor protein.

b) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 8 has significant sequence homology with aserine-threonine kinase from S. cerevisiae. An amino acid part-sequencederived therefreom (corresponding to nucleotides 1067 to 84 from SEQ IDNO: 8) with a part-sequence of the S. cerevisiae enzyme is depicted inFIG. 2. SEQ ID NO: 9 shows an N-terminally extended amino acidpart-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a serine-threonine kinase.

c) The amino acid sequence derived from the complementary strand to SEQID NO: 12 has significant sequence homology with a GTPase-activatingprotein from S. cerevisiae. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 475 to 353 from SEQ ID NO: 12)with a part-sequence of the S. cerevisiae protein is depicted in FIG.3A. A further amino acid part-sequence derived therefrom (correspondingto nucleotides 351 to 1 from SEQ ID NO: 12) with a part-sequence of theS. cerevisiae protein is depicted in FIG. 3B. SEQ ID NO: 13 and SEQ IDNO: 14 each show an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a GTPase-activating protein.

d) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 17 has significant sequence homology with a proteinfrom S. cerevisiae which is associated with a 5 r resistance tooverexpression of actin. An amino acid part-sequence derived therefrom(corresponding to nucleotides 933 to 157 from SEQ ID NO: 17) with apart-sequence of the S. cerevisiae protein is depicted in FIG. 4. SEQ IDNO: 18 shows an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a protein which has resistance to overexpression of actin.

e) The amino acid sequence derived from the coding strand to SEQ ID NO:21 has significant sequence homology with an Nuf1p-like protein from S.cerevisiae. An amino acid part-sequence derived therefrom (correspondingto nucleotides 117 to 794 from SEQ ID NO: 21) with a part-sequence ofthe S. cerevisiae protein is depicted in FIG. 5. SEQ ID NO: 22 shows anN-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of an Nuf1p-like protein.

f) The amino acid sequence derived from the coding strand to SEQ ID NO:26 has significant sequence homology with a calponin-homologous proteinfrom S. cerevisiae. An amino acid part-sequence derived therefrom(corresponding to nucleotides 438 to 767 from SEQ ID NO: 26) with apart-sequence of the S. cerevisiae protein is depicted in FIG. 6. SEQ IDNO: 27 shows an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a calponin-homologous protein.

g) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 30 has significant sequence homology with a proteinfrom C. maltosa which is essential for pseudohyphal development in C.maltosa. An amino acid part-sequence derived therefrom (corresponding tonucleotides 983 to 651 from SEQ ID NO: 30) with a part-sequence of theC. maltosa protein is depicted in FIG. 7A. Another amino acidpart-sequence derived therefrom (corresponding to nucleotides 661 to 596from SEQ ID NO: 30) with a part-sequence of the C. maltosa protein isdepicted in FIG. 7B. A third amino acid part-sequence derived therefrom(corresponding to nucleotides 591 to 1 from SEQ ID NO: 30) with apart-sequence of the C. maltosa protein is depicted in FIG. 7C. SEQ IDNO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 in each case show anN-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a protein which is essential for pseudohyphal development inC. maltosa.

h) The amino acid sequence derived from the coding strand to SEQ ID NO:36 has significant sequence homology with a protein from S. cerevisiaewhich interacts with actin. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 2 to 148 from SEQ ID NO: 36)with a part-sequence of the S. cerevisiae protein is depicted in FIG. 8.SEQ ID NO: 37 shows an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a protein which interacts with actin.

EXAMPLE 6

Isolation of Full-Length DNA

a) Construction of an A. gossypii Gene Library

High molecular weight cellular complete DNA from A. gossypii wasprepared from a 2-day old 100 ml culture grown in a liquid MA2 medium(10 g of glucose, 10 g of peptone, 1 g of yeast extract, 0.3 g ofmyo-inositol ad 1 000 ml). The mycelium was filtered off, washed twicewith distilled H₂O, suspended in 10 ml of 1M sorbitol, 20 mM EDTA,containing 20 mg of zymolyase 20T, and incubated at 27° C., shakinggently, for 30 to 60 min. The protoplast suspension was adjusted to 50mM Tris-HCl, pH 7.5, 150 mM NaCl, 100 mM EDTA and 0.5% strength sodiumdodecyl sulfate (SDS) and incubated at 65° C. for 20 min. After twoextractions with phenol/chloroform (1:1 vol/vol), the DNA wasprecipitated with isopropanol, suspended in TE buffer, treated withRNase, reprecipitated with isopropanol and resuspended in TE.

An A. gossypii cosmid gene library was produced by binding genomic DNAwhich had been selected according to size and partially digested withSau3A to the dephosphorylated arms of the cosmid vector Super-Cos1(Stratagene). The Super-Cos1 vector was opened between the two cos sitesby digestion with Xbal and dephosphorylation with calf intestinalalkaline phosphatase (Boehringer), followed by opening of the cloningsite with BamHl. The ligations were carried out in 20 μl, containing 2.5μg of partially digested chromosomal DNA, 1 μg of Super-Cos1 vectorarms, 40 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 0.5 mMATP and 2 Weiss units of T4-DNA ligase (Boehringer) at 15° C. overnight.The ligation products were packaged in vitro using the extracts and theprotocol of Stratagene (Gigapack II Packaging Extract). The packagedmaterial was used to infect E. coli NM554 (recA13, araD139,Δ(ara,leu)7696, Δ(lac)17A, galU, galK, hsrR, rps(str^(r)), mcrA, mcrB)and distributed on LB plates containing ampicillin (50 μg/ml).Transformants containing an A. gossypii insert with an average length of30-45 kb were obtained.

b) Storage and Screening of the Cosmid Gene Library

In total, 4×10⁴ fresh single colonies were inoculated singly into wellsof 96-well microtiter plates (Falcon, No. 3072) in 100 μl of LB medium,supplemented with the freezing medium (36 mM K₂HPO₄/13.2 mM KH₂PO₄, 1.7mM sodium citrate, 0.4 mM MgSO₄, 6.8 mM (NH₄)₂SO₄, 4.4% (wt/vol)glycerol) and ampicillin (50/μg/ml), allowed to grow at 37° C. overnightwith shaking, and frozen at −70° C. The plates were rapidly thawed andthen duplicated in fresh medium using a 96-well replicator which hadbeen sterilized in an ethanol bath with subsequent evaporation of theethanol on a hot plate. Before the freezing and after the thawing(before any other measures) the plates were briefly shaken in amicrotiter shaker (Infors) in order to ensure a homogeneous suspensionof cells. A robotic system (Bio-Robotics) with which it is possible totransfer small amounts of liquid from 96 wells of a microtiter plate tonylon membrane (GeneScreen Plus, New England Nuclear) was used to placesingle clones on nylon membranes. After the culture had been transferredfrom the 96-well microtiter plates (1920 clones), the membranes wereplaced on the surface of LB agar with ampicillin (50 μg/ml) in 22×22 cmculture dishes (Nunc) and incubated at 37° C. overnight. Before cellconfluence was reached, the membranes were processed as described byHerrmann, B. G., Barlow, D. P. and Lehrach, H. (1987) in Cell 48, pp.813-825, including as additional treatment after the first denaturationstep a 5-minute exposure of the filters to vapors on a pad impregnatedwith denaturation solution on a boiling water bath.

The random hexamer primer method (Feinberg, A. P. and Vogelstein, B.(1983), Anal. Biochem. 132, pp.6-13) was used to label double-strandedprobes by uptake of [alpha-³²P]dCTP with high specific activity. Themembranes were prehybridized and hybridized at 42° C. in 50% (vol/vol)formamide, 600 mM sodium phosphate, pH 7.2, 1 mM EDTA, 10% dextransulfate,1% SDS, and 10× Denhardt's solution, containing salmon sperm DNA(50 μg/ml) with ³²P-labeled probes (0.5-1×10⁶cpm/ml) for 6 to 12 h.Typically, washing steps were carried out at 55 to 65° C. in 13 to 30 mMNaCl, 1.5 to 3 mM sodium citrate, pH 6.3, 0.1 % SDS for about 1 h andthe filters were autoradiographed at −70° C. with Kodak intensifyingscreens for 12 to 24 h. To date, individual membranes have been reusedsuccessfully more than 20 times. Between the autoradiographies, thefilters were stripped by incubation at 95° C. in 2 mM Tris-HCI, pH 8.0,0.2 mM EDTA, 0.1% SDS for 2×20 min.

c) Recovery of Positive Colonies from the Stored Gene Library

Frozen bacterial cultures in microtiter wells were scraped out usingsterile disposable lancets, and the material was streaked onto LB agarPetri dishes containing ampicillin (50 μg/ml). Single colonies were thenused to inoculate liquid cultures to produce DNA by the alkaline lysismethod (Birnboim, H. C. and Doly, J. (1979), Nucleic Acids Res. 7,pp.1513-1523).

d) Full-Length DNA

It was possible as described above to identify clones which harbor aninsert with the appropriate complete sequence. These clones have theinternal names:

“Oligo 8v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 4. The protein encoded therebypreferably comprises at least one of the amino acid sequences as shownin SEQ ID NO: 5, 6 and 7.

“Oligo 25/39v”. The insert comprising the complete sequence has anucleic acid sequence as shown in SEQ ID NO: 10.

“Oligo 46v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 15.

“Oligo 103v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 19.

“Oligo 128v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 23. The protein encoded therebypreferably comprises at least one of the amino acid sequences as shownin SEQ ID NO: 24 and 25.

“Oligo 150v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 28.

“Oligo 177v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 34.

“Oligo 145v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 38. TABLE 1 Sequence survey SEQ IDDescription of the Sequence NO: Oligo sequence homology 1 008 DNApart-sequence Cell wall pre- 2 008 Amino acid part-sequence cursorprotein derived from the com- Cwp 1 from S. plementary strand tocerevisiae SEQ ID NO: 1 3 008 Amino acid part-sequence derived from thecom- plementary strand to SEQ ID NO: 1 4 008 DNA full-length sequence 5008 Amino acid sequence cor- responding to the coding region of SEQ IDNO: 4 from position 523 to 996 6 008 Amino acid sequence cor- respondingto the coding region of SEQ ID NO: 4 from position 1523 to 2035 7 008Amino acid sequence cor- responding to the coding region of SEQ ID NO: 4from position 2222 to 2425 8 025/ DNA part-sequence Serine-threonine 039protein kinase from S. cerevisiae 9 025/ Amino acid part-sequence 039derived from the com- plementary strand to SEQ ID NO: 8 10 025/ DNAfull-length sequence 039 11 025/ Amino acid sequence cor- 039 respondingto the coding region of SEQ ID NO: 10 from position 821 to 3703 12 046DNA part-sequence GTPase-activat- 13 046 Amino acid part-sequence ingprotein from derived from the com- S. cerevisiae plementary strand toSEQ ID NO: 12 14 046 Amino acid part-sequence derived from the com-plementary strand to SEQ ID NO: 12 15 046 DNA full-length sequence 16046 Amino acid sequence cor- responding to the coding region of SEQ IDNO: 15 from position 314 to 3556 17 103 DNA part-sequence Protein which18 103 Amino acid part-sequence has resistance derived from the com- tooverexpres- plementary strand to sion of actin or SEQ ID NO: 17contributes to 19 103 DNA full-length sequence this resistance 20 103Amino acid sequence cor- from S. responding to the coding cerevisiaeregion of SEQ ID NO: 19 from position 584 to 1441 21 128 DNApart-sequence Nuf1p-like pro- 22 128 Amino acid part-sequence tein fromS. derived from the coding cerevisiae strand to SEQ ID NO: 21 23 128 DNAfull-length sequence 24 128 Amino acid sequence cor- responding to thecoding region of SEQ ID NO: 23 from position 272 to 703 25 128 Aminoacid sequence cor- responding to the coding region of SEQ ID NO: 23 fromposition 775 to 1374 26 150 DNA part-sequence Calponin- 27 150 Aminoacid part-sequence homologous derived from the coding protein fromstrand to S. cerevisiae SEQ ID NO: 26 28 150 DNA full-length sequence 29150 Amino acid sequence cor- responding to the coding region of SEQ IDNO: 28 from position 628 to 1227 30 177 DNA part-sequence Protein is es-31 177 Amino acid part-sequence sential for derived from the com-pseudohyphal plementary strand to development in SEQ ID NO: 30 Candidamaltosa 32 177 Amino acid part-sequence derived from the com- plementarystrand to SEQ ID NO: 30 33 177 Amino acid part-sequence derived from thecom- plementary strand to SEQ ID NO: 30 34 177 DNA full-length sequence35 177 Amino acid sequence cor- responding to the coding region of SEQID NO: 34 from position 768 to 2366 36 145 DNA part-sequence Proteinfrom 37 145 Amino acid part-sequence S. cerevisiae derived from thecoding which interacts strand to with actin SEQ ID NO: 36 38 145 DNAfull-length sequence 39 145 Amino acid sequence cor- responding to thecoding region of SEQ ID NO: 38 from position 735 to 2336

1. An isolated polynucleotide that can be isolated from Ashbya gossypiiand that codes for a protein associated with construction of a cell wallor a cytoskeleton of an organism.
 2. The polynucleotide of claim 1,which has a structural or functional property of a protein selected fromthe group consisting of a cell wall protein, a serine-threonine proteina GTPase-activating protein, a protein that has resistance to overexpression of actin or contributes to such resistance, a Nuf1p-likeprotein, a calponin-homologous protein, a protein that is essential forpseudohyphal development, and a protein that interacts with actin. 3.The polynucleotide of claim 1, comprising: the nucleic acid sequence ofSEQ ID NO: 1, 8, 12, 17, 21, 26, 30 or 36 a sequence complementarythereto; or a sequence derived from said nucleic acid sequence or saidsequence complementary thereto through degeneracy of the genetic code.4. The polynucleotide of claim 1, which comprises a nucleic acid thatcontains the sequence of SEQ ID NO: 4, 10, 15, 19, 23, 28, 34 or 38, ora fragment thereof.
 5. An oligonucleotide that hybridizes to thepolynucleotide of claim
 1. 6. An isolated polynucleotide that hybridizesto the oligonucleotide of claim 5, and codes for a gene product derivedfrom a microorganism of the genus Ashbya or a functional equivalentthereof.
 7. An isolated polypeptide is encoded by the polynucleotide ofclaim 1 or a fragment thereof.
 8. An expression cassette comprising thepolynucleotide of claim 1 operatively linked to at least one regulatorysequence.
 9. A recombinant vector comprising at least one expressioncassette of claim
 8. 10. A prokaryotic or eukaryotic host celltransformed with at least one vector of claim
 9. 11. The host cell ofclaim 10, wherein functional expression of said protein is modulated.12. A The host cell of claim 10, which is a microorganism of the genusAshbya.
 13. A method for microbiological production of vitamin B2 or aprecursor or derivative thereof comprising expressing the polynucleotideof claim 1 in a microorganism.
 14. A method for recombinant productionof the polypeptide of claim 7 comprising expressing said polynucleotidein a microorganism.
 15. A method for detecting an effector target formodulating microbiological production of vitamin B2 or a precursor orderivative thereof comprising treating a microorganism capable of themicrobiological production of said vitamin B2 or the precursor orderivative thereof with an effector that interacts with a target whereinsaid target comprises the polypeptide of claim 7 or a nucleic acid thatencodes said polypeptide and detecting said effector target.
 16. Amethod for modulating microbiological production of vitamin B2 or aprecursor or derivative thereof comprising treating a microorganismcapable of the microbiological production of said vitamin B2 or theprecursor or derivative thereof with an effector that interacts with atarget wherein said target comprises the polypeptide of claim 7 or anucleic acid that encodes said polypeptide.
 17. An isolated effectorselected from the group consisting of: antibodies or antigen-bindingfragments thereof that bind to the polypeptide of claim 7; polypeptideligands that are different from said antibodies or antigen-bindingfragments and that interact with said polypeptide; low molecular weighteffectors that modulate a biological activity of said polypeptide;antisense nucleic acid sequences, catalytic RNA molecules and ribozymeswhich interact with a nucleic acid sequence that encodes saidpolypeptide; and combinations and mixtures thereof.
 18. A method formicrobiological production of vitamin B2 or a precursor or derivativethereof comprising: culturing the host cell of claim 10 under conditionsfavoring the production of vitamin B2 or the precursor or derivativethereof; and isolating a desired product.
 19. The method of claim 18,wherein the host cell is treated with an effector before or duringculturing.
 20. The method of claim 18, wherein the host cell is amicroorganism of the genus Ashbya.
 21. A method for modulatingproduction of vitamin B2 or a precursor or derivative thereof in amicroorganism of the genus Ashbya comprising treating said microorganismwith the polynucleotide of claim
 1. 22. A method for modulatingproduction of vitamin B2 or a precursor or derivative thereof in amicroorganism of the genus Ashbya comprising treating said microorganismwith the polypeptide of claim
 7. 23. A method for modulatingconstruction of a cell wall or cytoskeleton of a microorganism of thegenus Ashbya comprising culturing said microorganism for microbiologicalproduction of vitamin B2 or a precursor or derivative thereof with thepolynucleotide of claim 1 or with a polypeptide encoded by saidpolynucleotide.
 24. The host of claim 12, which has a modified cell wallor cytoskeleton construction as compared with a non-transformed cell,wherein said modified cell wall or cytoskeleton construction providesfor an increased production of vitamin B2 or a precursor or derivativethereof.
 25. The polynucleotide of claim 1, wherein the organism is A.gossypii, S. cerevisiae, or C. maltosa.
 26. The polynucleotide of claim1, wherein the protein is associated with a developmental-specific orenvironmentally-related change to morphology of the organism.
 27. Thepolynucleotide of claim 2, wherein the protein is derived from amicroorganism of A. gossypii, S. cerevisiae, or C. maltosa.
 28. Theoligonucleotide of claim 5, wherein hybridization is under stringentconditions.
 29. The polynucleotide of claim 6, wherein hybridization isunder stringent conditions.
 30. An isolated polypeptide or fragmentthereof encoded by the polynucleotide of claim
 6. 31. An isolatedpolypeptide or fragment thereof which has an amino acid sequence thatcomprises at least ten consecutive amino acid residues of SEQ ID NO: 2,3, 5, 6, 7, 9, 11, 13, 14, 16, 18, 20, 22, 24, 25, 27, 29, 31, 32, 33,35, 37 or 39, or a functional equivalent thereof.
 32. The polypeptide ofclaim 31, which has an activity comparable with a protein selected fromthe group consisting of a cell wall protein, a serine-threonine protein,a GTPase-activating protein, a protein that has resistance to overexpression of actin or contributes to such resistance, a Nuf1p-likeprotein, a calponin-homologous protein, a protein that is essential forpseudohyphal development, and a protein that interacts with actin. 33.The polypeptide of claim 32, wherein the protein is derived from amicroorganism of A. gossypii, S. cerevisiae, or C. maliosa.
 34. The hostcell of claim 10, wherein biological activity of said protein is reducedor increased.
 35. The method of claim 11, wherein modulating comprisesan increase or decrease in the functional expression of said protein.36. The method of claim 13, wherein expressing said polypeptide resultsin an improved production of vitamin B2 or a precursor or derivativethereof by said microorganism.
 37. The method of claim 36, wherein theimproved production comprises an increased yield, production orefficiency of production by said microorganism.
 38. The method of claim15, wherein detecting validates said effector target.
 39. The method ofclaim 15, wherein the effector binds to said target.
 40. The method ofclaim 15, further comprising isolating said target.
 41. The method ofclaim 19, wherein the effector is selected from the group consisting of:antibodies or antigen-binding fragments thereof that bind to apolypeptide associated with construction of a cell wall or acytoskeleton of an organism; polypeptide ligands that are different fromsaid antibodies or antigen-binding fragments and that interact with saidpolypeptide; low molecular weight effectors that modulate a biologicalactivity of said polypeptide; antisense nucleic acid sequences,catalytic RNA molecules and ribozymes which interact with a nucleic acidsequence that encodes said polypeptide; and combinations and mixturesthereof.
 42. The method of claim 21, wherein modulating comprises anincrease in rate or amount of the vitamin B2 or the precursor orderivative thereof produced by said microorganism.
 43. The method ofclaim 22, wherein modulating comprises an increase in rate or amount ofthe vitamin B2 or the precursor or derivative thereof produced by saidmicroorganism.
 44. A recombinant cell with a modified cell wall orcytoskeleton construction that provides for an increased production ofvitamin B2 or a precursor or derivative thereof as compared with anon-recombinant cell.
 45. The recombinant cell of claim 44, which is A.gossypii, S. cerevisiae, or C. maltosa.